Biomedical subjects

H Niemann

Publications and source records attributed to H Niemann.

At least 37 records · Page 2Linked to original sources

PubMed2003

The transgenic rabbit as model for human diseases and as a source of biologically active recombinant proteins.

Until recently, transgenic rabbits were produced exclusively by pronuclear microinjection which results in additive random insertional transgenesis; however, progress in somatic cell cloning based on nuclear transfer will soon make it possible to produce rabbits with modifications to specific genes by the combination of homologous recombination and subsequent prescreening of nuclear donor cells. Transgenic rabbits have been found to be excellent animal models for inherited and acquired human diseases including hypertrophic cardiomyopathy, perturbed lipoprotein metabolism and atherosclerosis. Transgenic rabbits have also proved to be suitable bioreactors for the production of recombinant protein both on an experimental and a commercial scale. This review summarizes recent research based on the transgenic rabbit model.

Animals↗

PubMed2003

Advances in biotechnology: new tools in future pig production for agriculture and biomedicine.

Biotechnology in livestock comprises an arsenal of reproductive biotechniques and molecular genetics. While molecular genetics are poorly developed in swine, reproductive techniques are more advanced and applied under field conditions. This review describes three selected examples of our own research to illustrate the implication of biotechnology in future pig reproduction. Sperm sexing technology is now available and can be used to generate piglets of the desired sex by IVF and ICSI. First studies also indicate satisfactory success rates following intrauterine insemination with sexed spermatozoa. Cloning technique and production of transgenic pigs require information about the regulation and time course of gene expression during in vitro production and pre-implantation development. Information on gene expression is scare in porcine embryos. With the exception of transcripts for the oestrogen receptor gene, no mRNA's from the activated porcine genome have been identified. Recent development of cDNA arrays might help to identify a larger amount of genes in single embryos. Remarkable progress has been made in organ transplantation technology. As the demand for human organs is increasing rapidly, the pig might serve as donor of xenotranplants, provided the transmission of zoonoses from the donor animal to the human recipient is prevented, donor organ anatomy and function are compatible, and immunological rejections (HAR, VAR MAC) can be overcome. The most promising strategy is the synthesis of human complement regulatory proteins in the pig. Transgenic pigs have been generated for hDAF or hCD 46 and their hearts have been transplanted into non human primates for up to 90 days. HCD 59 driven by CMV promotor provides significant protection against HAR at least under in vitro and in vivo conditions. Current studies indicate a temporary use in patients within the next 3-5 years. As the microinjection technology to produce transgenic offspring is time consuming and very expensive, nuclear transfer technology provides a possibility for multiplication without going through the germ line with recombination effects. A functional nuclear transfer system will be crucial for xenotransplantation as it is anticipated that the expression of several transgenes will be required.

Agriculture↗

PubMed2003

Expression of active human blood clotting factor VIII in mammary gland of transgenic rabbits.

Human clotting factor VIII is probably the largest protein to be expressed to date in the mammary gland of a transgenic animal, and it requires extensive posttranslational modification to achieve full biological activity. The mammary gland specific construct mWAP-hFVIII-MT-I was injected into the pronuclei of rabbit zygotes, and three transgenic offspring were obtained. Founder 385 showed germ-line transmission of a single integrated copy, and a homozygous line was established from this animal. The rhFVIII was transcribed and translated exclusively in the mammary gland. The activity of rhFVIII in the rabbit milk ranged from 5 to 8% of that found in normal human plasma. Results indicate the suitability of the transgenic rabbit mammary gland for rhFVIII production.

Animals↗

PubMed2003

Mitogenic and anti-apoptotic activity of insulin on bovine embryos produced in vitro.

Insulin improves development of mammalian preimplantation embryos and, in addition to the regulation of glucose transport, it exerts mitogenic and anti-apoptotic activities. The expression of glucose transporters (Glut) mediating the uptake of this essential energy substrate is critical for embryo survival. An impaired expression of Glut leads to an increase in apoptosis at the blastocyst stage and involves Bax. The various effects of insulin were unravelled by supplementing the in vitro culture medium with insulin (1.7 micromol l(-1)) and (i) the rates of cleavage and blastocyst development were recorded; (ii) mitogenic activity was studied by determining the total number of blastocyst cells and the ratio between trophectoderm and inner cell mass (ICM) cells; (iii) the frequency of apoptosis in blastocysts was determined by the TdT-mediated duTP nick-end labelling (TUNEL) assay and by quantification of the relative amounts of mRNA for Bax and Bcl-XL; and (iv) expression for Glut1, Glut3 and Glut8 transcripts was compared between embryos cultured in the presence or absence of insulin. Insulin increased rates of cleavage (81.2+/-2.2 (control) to 86.0+/-2.5) and blastocyst development (24.7+/-1.9 to 31.3+/-1.2), and number of blastocyst cells (123.7+/-6.0 to 146.3+/-6.6); the increase in the number of blastocyst cells was due to a significantly higher number of trophectoderm cells (82.3+/-5.0 versus 100.3+/-5.5). Blastocysts derived from cultures supplemented with insulin showed a significant decrease in apoptosis as determined by the TUNEL assay (14.8+/-0.9 to 12.2+/-0.7). No effects of insulin on the mRNA expression of Glut isoforms and Bax and Bcl-XL were found. These results demonstrate that the mitogenic and anti-apoptotic effects of insulin on bovine preimplantation embryos did not correlate with changes in the amounts of mRNA for the glucose transporter isoforms Glut1, -3 and -8, or transcripts for Bax and Bcl-XL.

Animals↗

PubMed2002

Effects of in vivo prematuration and in vivo final maturation on developmental capacity and quality of pre-implantation embryos.

In current in vitro production (IVP) systems, oocytes lack in vivo dominant and preovulatory follicular development, which may compromise pregnancy and viability of calves born. When an oocyte sets off in vivo on the road toward fertilization, it contains numerous transcripts and proteins necessary to survive the first few cell cycles of embryonic development. It is not yet known during which period of development the oocyte builds up the store, possibly primarily during the major growth phase of the oocyte, which is completed at the time a follicle reaches the size of 3 mm. Here, we investigated to what extent the later phases of follicular development, such as prematuration in the dominant follicle before the LH surge and ensuing final maturation in the preovulatory follicle, contribute to oocyte competence and development into viable biastocysts. Recent studies on in vivo vs in vitro oocyte maturation employed oocytes from an identical preovulatory development by applying ovum pick-up (OPU) twice (before and 24 h after the LH surge) in each cow treated for superovulation with a controlled LH surge. The embryo recovery rates at Day 7 of IVC after IVF were similar: 44% (97/219) for in vivo- vs 41% (87/213) for in vitro-matured oocytes, which shows that the natural environment during final maturation is not essential for the mere in vitro development of the prematured oocyte beyond the 8- to 16-cell stage. However, in vivo maturation appeared to contribute to the oocyte's quality in a more subtle way, as indicated by a significant increase in the proportion of expanded blastocysts and a more physiological degree of chromosome aberrations of the embryos. In blastocysts derived from in vivo-matured oocytes, 21% of the embryos were mixoploid vs 50% from in vitro-matured oocytes, concomitant with a higher number of cells (96 vs 54 per normal blastocyst). The expression pattern of a set of six developmentally important genes was, however, not significantly altered in blastocysts derived from in vivo-matured oocytes. Certain deviations were observed compared with the levels of entirely in vivo-developed control blastocysts, which suggests that the beneficial effects of in vivo maturation are possibly exerted at initial stages of embryonic development. Prematuration in vivo, occurring in a dominant follicle developing from about 8 mm into the preovulatory follicle, is accompanied by changes in protein synthesis of the cumulus oocyte complex (COC). Presumably, the differentially expressed proteins are involved in equipping the oocyte with further developmental competence. Although we have unraveled some important biochemical and cellular biological features of the oocyte, further research on in vivo processes is essential to improve in vitro embryo production in practice.

Animals↗

PubMed2002

Gene expression patterns in bovine in vitro-produced and nuclear transfer-derived embryos and their implications for early development.

Bovine in vitro-produced (IVP) and nuclear transfer (NT)-derived embryos differ from their in vivo-developed counterparts in a number of characteristics. A preeminent observation is the occurrence of the large offspring syndrome, which is correlated with considerable embryonic fetal and postnatal losses. We summarize here results from our studies in which we compared gene expression patterns from IVP and NT-derived embryos with those from their IVP counterparts. Numerous aberrations were found in IVP and NT-derived embryos, including a complete lack of expression, an induced expression, or a significant up- or downregulation of a specific gene. These alterations may affect a number of physiological functions and are considered as a kind of stress response of the embryos to deficient environmental conditions. We hypothesize that the alterations are caused by epigenetic modifications, primarily by changes in the methylation patterns. Unravelling these epigenetic modifications is promising to reveal the underlying mechanisms of the large offspring syndrome.

Animals↗

PubMed2002

In vitro production and nuclear transfer affect dosage compensation of the X-linked gene transcripts G6PD, PGK, and Xist in preimplantation bovine embryos.

Equal expression of X-linked genes such as G6PD and PGK in females and males and the initiation of X-chromosome inactivation are critically dependent on the expression of the X-inactive specific transcript (Xist). The objective of the present study was to determine the effects of in vitro production (IVP) and nuclear transfer (NT) on the relative abundance (RA) of the X-linked transcripts G6PD, PGK, and Xist in preimplantation bovine embryos. In experiment 1, sex-determined IVP or in vivo-produced embryos were analyzed for mRNA expression of the 3 genes. The sex ratio was 36% vs. 64% in IVP blastocysts and thus deviated significantly from the expected ratio of 50% in the vivo control group. The RA of G6PD transcripts was significantly higher in female IVP embryos than in male embryos. In contrast, no significant differences were seen between in vivo-derived female embryos and their male counterparts. At the morula stage, female IVP embryos transcribed significantly more PGK mRNA than did male embryos. However, blastocysts did not exhibit significant differences in PGK transcripts. No differences were observed for in vivo-derived embryos with regard to the RA of PGK transcripts. The RA of Xist mRNA was significantly higher in all female embryos than in their male counterparts. In experiment 2, IVP, in vivo-developed, NT-derived, and parthenogenetic embryos carrying two X chromosomes of either maternal and paternal origin or of maternal origin only (parthenogenotes) were analyzed for the RA of the 3 genes. In NT-derived morulae, the RA of G6PD transcripts was significantly increased compared with their IVP and in vivo-generated counterparts. G6PD transcript levels were significantly increased in IVP blastocysts compared with in vivo-generated and parthenogenetic embryos. At the morula stage, PGK transcripts were similar in all groups, but the RA of PGK transcripts was significantly higher in IVP blastocysts than in their in vivo-generated, parthenogenetic, and NT-derived counterparts. The RA of Xist was significantly elevated in NT-derived morulae compared with IVP, in vivo-generated, and parthenogenetic embryos. NT-derived blastocysts showed an increased Xist expression compared with that of IVP, in vivo-generated, and parthenogenetic embryos. Results of the present study show for the first time that differences in X-chromosome-linked gene transcript levels are related to a perturbed dosage compensation in female and male IVP and female NT-derived embryos. This finding warrants further studies to improve IVP systems and NT protocols to ensure the production of embryos with normal gene expression patterns.

Animals↗

PubMed2002

Application of cDNA arrays to monitor mRNA profiles in single preimplantation mouse embryos.

Array technology is a widely used tool for gene expression profiling in various biological systems. However, the application of this method to mammalian preimplantation embryos is limited by the small amount of mRNA that can be extracted from a single embryo, which is not sufficient for array analysis. Here we report a protocolfor the rapid global amplification of embryonic mRNA that permits the generation of expression profiles from single murine blastocysts. The approach combines global PCR and 77 RNA polymerase amplification and allows the preparation of labeled, amplified RNA for array hybridization from single murine blastocysts containing approximately 1.5 pg mRNA in less than 12 h. We demonstrate that this amplification procedure is highly reproducible and does not bias original relative mRNA levels. Signal patterns from various embryonic stages of murine development revealed marked differences in mRNA expression that were in accordance with previously published data. We found genes known to be involved in embryonic apoptosis expressed at different levels in individual murine day 3.5 blastocysts. This technique can thus be used to assess embryonic viability and investigate molecular mechanisms of embryonic development.

Animals↗

PubMed2002

Effects of oocyte maturation regimen on the relative abundance of gene transcripts in bovine blastocysts derived in vitro or in vivo.

Bovine embryos produced in vitro differ substantially from embryos produced in vivo in the mRNA expression patterns of genes important for development. Several factors in the in vitro production systems have profound effects on embryonic mRNA expression patterns. The effects of the type of maturation on the expression pattern of genes important for development in blastocysts produced in vitro have not yet been investigated. The aim of the present study was to investigate the effects of various maturational protocols on the relative abundance of a panel of six marker genes, indicative of compaction and cavitation, metabolism, stress susceptibility and RNA processing, in bovine blastocysts produced in vitro. Four groups of blastocysts were analysed by a sensitive semi-quantitative RT-PCR assay. Blastocysts were produced in vitro from oocytes of different origin from: (1) 3-8 mm follicles; (2) preovulatory follicles before the LH surge; and (3) preovulatory follicles 24 h after the LH surge. The first two groups were matured in vitro, whereas the third group had undergone maturation in vivo. A fourth group comprised blastocysts developed entirely in vivo. Expression of glucose transporter 1 was significantly (P < 0.05) higher, and expression of desmocollin 2 and plakophilin tended to be higher (P < 0.1) for in vivo (group 4) compared with in vitro blastocysts (group 1), whereas no differences were found for heat shock protein 70.1, E-cadherin and poly(A) polymerase. Expression of the six transcripts did not differ among blastocysts produced in vitro from oocytes of groups 1, 2 and 3. Results indicate that alterations in the relative abundance of these transcripts in blastocysts produced in vitro cannot primarily be attributed to the origin of the oocyte, but are likely to have been induced by post-maturation or fertilization culture conditions.

Animals↗

PubMed2001

Cytomegalovirus early promoter induced expression of hCD59 in porcine organs provides protection against hyperacute rejection.

The critical shortage of human donor organs has generated growing interest for porcine to human xenotransplantation. The major immunological barrier to xenotransplantation is the hyperacute rejection (HAR) response that is mediated by preformed xenoreactive antibodies and complement. A promising strategy to control the complement activation, is the expression of human complement regulatory proteins in transgenic animals. We have used the human early cytomegalovirus (CMV) promoter to drive expression of the human complement regulatory protein CD59 (hCD59) in transgenic pigs. A total of eight live transgenic founder animals was born from which five transgenic lines could be established. mRNA analysis and Western blotting revealed high expression of hCD59 in heart, kidney, skeletal muscle, and skin in animals of lines 1 and 5, as well as in the pancreas of four lines. This pattern of expression was confirmed by immunhistological staining. A cell-specific expression in heart and kidney tissue of transgenic lines 1 and 5 was determined. Primary fibroblasts and endothelial cell cultures derived from the aorta of transgenic pigs showed a significantly diminished sensitivity against the challenge with xenoreactive human antibodies and complement whereas non-transgenic control cells were highly susceptible to complement mediated lysis. Ex vivo perfusion of kidneys with pooled human blood revealed a significant protective effect of hCD59 against HAR. The average survival of transgenic kidneys was significantly extended (P<0.05) over nontransgenic controls (207.5+/-54.6 vs. 57.5+/-64.5 min). These data support the concept that hCD59 protects nonprimate cells against human complement mediated lysis and suggest that donor pigs transgenic for hCD59 could play a crucial role in clinical xenotransplantation. Two of five hCD59 transgenic lines showed strong hCD59 expression in several organs relevant for xenotransplantation and a protective effect against HAR. This indicates that the use of the CMV-promoter can facilitate the selection process for optimized transgene expression.

3T3 Cells↗

PubMed2001

Progress in reproductive biotechnology in swine.

This article summarizes recent progress in reproductive biotechnology in swine with special reference to in vitro production of embryos, generation of identical multiples, and transgenic pigs useful for xenotransplantation. In vitro production (in vitro maturation, in vitro fertilization, and in vitro culture) of viable porcine embryos is possible, although with much lower success rates than in cattle. The main problems are insufficient cytoplasmic maturation of porcine oocytes, a high proportion of polyspermic fertilization and a low proportion of blastocysts that, in addition, are characterized by a low number of cells, hampering their development in vivo upon transfer to recipients. Microsurgical bisection of morula and blastocyst stage embryos leads to a 2 to 3% monozygotic twinning rate of the transferred demiembryos, which is similar to that in rabbits and mice but considerably lower than in ruminants. It was found that with decreasing quality an increasing proportion of demi-embryos did not possess an inner cell mass. Porcine individual blastomeres derived from 4- and 8-cell embryos can be cultured in defined medium to the blastocyst stage. Leukemia inhibitory factor has been shown to be effective at defined embryonic stages and supports the formation of the inner cell mass in cultured isolated blastomeres in a concentration-dependent manner. For maintaining pregnancies with micromanipulated porcine embryos, it is not necessary to transfer extraordinarily high numbers of embryos. Porcine nuclear transfer is still struggling from the inefficiency of producing normally functioning blastocysts. Blastomeres, blastocyst-derived cells, fibroblasts and granulosa cells have been employed as donor cells in porcine nuclear transfer and have yielded blastocysts. Recently, the generation of the first piglets from somatic cell nuclear transfer has been achieved. DNA-microinjection into pronuclei of porcine zygotes has reliably resulted in the generation of transgenic pigs, which have special importance for the production of valuable pharmaceutical proteins in milk and xenotransplantation. It has been demonstrated that by expression of human complement regulatory proteins in transgenic pigs the hyperacute rejection response occurring after xenotransplantation can be overcome in a clinically relevant manner. Although biotechnological procedures in swine have recently undergone tremendous progress, the development is still lagging behind that in cattle and sheep. With regard to genetic engineering, considerable progress will originate from the possibility of employing homologous recombination in somatic cell lines and their subsequent use in nuclear transfer. In combination with the increasing knowledge in gene sequences this will allow in the foreseeable future widespread use in the pig industry either for agricultural or biomedical purposes.

Animals↗

PubMed2001

Glucose transporter expression is developmentally regulated in in vitro derived bovine preimplantation embryos.

Glucose is readily been taken up and utilized by preimplantation embryos from different species. However, a comprehensive analysis of the glucose transporter expression throughout preimplantation development is still missing. Here, we have investigated the expression of facilitative glucose transporters (Glut1-5 and 8) and sodium-dependent-glucose transporter (SGLT-I) in bovine oocytes and preimplantation embryos up to d16 of development, using RT-PCR and immunohistochemistry. The embryos were produced in vitro by IVM-IVF. Glut1, Glut3, Glut8, and SGLT-I were expressed in all stages studied. Glut4 transcripts were first detected at the blastocyst stage. Glut2 expression was restricted to the period of blastocyst elongation at d14 and d16. Transcription of the fructose transporter Glut5 started at the 8-/16-cell stage. Our results show a distinct expression pattern for glucose transporters during bovine embryo development in vitro indicating specialized functions for these isoforms at different developmental stages in bovine embryos. Mol. Reprod. Dev. 60:370-376,

Animals↗

PubMed2001

Identification and analysis of eukaryotic promoters: recent computational approaches.

The DNA sequence of several higher eukaryotes is now complete, and we know the expression patterns of thousands of genes under a variety of conditions. This gives us the opportunity to identify and analyze the parts of a genome believed to be responsible for most transcription control--the promoters. This article gives a short overview of the state-of-the-art techniques for computational promoter localization and analysis, and comments on the most recent advances in the field.

Base Sequence↗

PubMed2001

Joint modeling of DNA sequence and physical properties to improve eukaryotic promoter recognition.

We present an approach to integrate physical properties of DNA, such as DNA bendability or GC content, into our probabilistic promoter recognition system McPROMOTER. In the new model, a promoter is represented as a sequence of consecutive segments represented by joint likelihoods for DNA sequence and profiles of physical properties. Sequence likelihoods are modeled with interpolated Markov chains, physical properties with Gaussian distributions. The background uses two joint sequence/profile models for coding and non-coding sequences, each consisting of a mixture of a sense and an anti-sense submodel. On a large Drosophila test set, we achieved a reduction of about 30% of false positives when compared with a model solely based on sequence likelihoods.

Animals↗

PubMed2001

Effects of culture system and protein supplementation on mRNA expression in pre-implantation bovine embryos.

Assisted reproduction technologies have made great progress during the last 15 years in most mammalian species, including humans. Growing evidence indicates that bovine pre-implantation development is a superior model for investigating early human development than the mouse. The purpose of this study was to investigate the effects of two basic culture systems [tissue culture medium (TCM) with 5% CO(2) in air or synthetic oviduct fluid (SOF) with 7% O(2), 88% N(2,) 5% CO(2)] and various protein supplements (serum, bovine serum albumin or polyvinyl alcohol) on the relative abundance of a set of developmentally important gene transcripts in bovine morulae and blastocysts and to compare the results with those for their in-vivo-derived counterparts. The basic culture system including the basic medium composition and oxygen tension had profound effects on the amounts of specific transcripts in bovine embryos, whereas the 'protein source' had only weak effects. Significant differences (P < or = 0.05) in the relative abundance of specific gene transcripts were detected between in-vivo and in-vitro-derived embryos, especially at the morula stage. More differences were found between embryos produced in the TCM system and in-vivo-derived embryos than between SOF-generated embryos and their in-vivo counterparts. No differences were found in the relative abundance of gene transcripts in embryos generated under chemically defined conditions in the two different laboratories. It is concluded that the SOF system provides an environment in which pre-implantation development of bovine embryos is more similar to that occurring in vivo than in the TCM system.

Animals↗

PubMed2001

Nuclear transfer protocol affects messenger RNA expression patterns in cloned bovine blastocysts.

The successful production of embryos by nuclear transfer (NT) employing cultured somatic donor cells depends upon a variety of factors. The objective of the present study was to investigate the effects 1) of two different activation protocols, 2) the use of quiescent or nonquiescent donor cells (G(0) or G(1) of the cell cycle), and 3) passage number of donor cells on the relative abundance (RA) of eight specific mRNAs (DNA methyltransferase, DNMT; mammalian achaete-scute homologue, Mash2; glucose transporter-1, Glut-1; heat shock protein 70.1, Hsp; desmocollin II, Dc II; E-cadherin, E-cad; interferon tau, IF; insulin-like growth factor 2 receptor, Igf2r) in single blastocysts employing a semiquantitative reverse transcription-polymerase chain reaction assay. The results were compared with those for their in vitro (IVP)- and in vivo-generated noncloned counterparts. In experiment 1, employing either FBA (fusion before activation) or AFS (fusion and activation simultaneously) to generate NT blastocysts, Hsp mRNAs were not found in NT embryos from either protocol, whereas Hsp transcripts were detectable in IVP embryos. The relative abundance (RA) of IF transcripts was significantly increased in the AFS and IVP groups compared to the FBA treatment. In experiment 2, the use of either G(0) or G(1) donor cells to produce cloned embryos both significantly reduced the relative amount of DNMT transcripts and significantly increased the RA of Mash2 compared to the IVP embryos. In addition, IF transcript levels were significantly elevated in NT blastocysts employing G(1) donor cells for NT compared to IVP embryos and those generated using G(0) cells. In experiment 3, donor cells, either from passsage 5/6 or 8, were employed for NT. DNMT transcripts were significantly decreased, whereas Mash2 transcripts were significantly increased in both NT groups compared to their IVP counterparts. The amount of IF mRNA was significantly higher in P8-derived than in P5/6 and IVP embryos. In experiment 4, the RA of DNMT transcripts was decreased in in vivo-derived blastocysts compared to those produced in vitro. Mash2 expression was increased in in vivo embryos and those IVP embryos produced in medium containing Sigma BSA. The RA of Hsp was higher in IVP embryos produced in serum containing medium than in those produced in Sigma BSA or in vivo. In vivo embryos and those produced in Life Technologies BSA had the lowest expression of IF transcripts. Expression of all other genes was not affected by variation in NT methodology or IVP culture systems throughout experiments 1-4. In conclusion, depending on steps of the cloning procedure NT-derived embryos display marked differences from their IVP- and in vivo-derived counterparts. An aberrant expression pattern in NT embryos was found with respect to genes thought to be involved in stress adaptation, trophoblastic function, and DNA methylation during preimplantation development.

Animals↗

PubMed2001

Changes in the relative abundance of mRNA transcripts for insulin-like growth factor (IGF-I and IGF-II) ligands and their receptors (IGF-IR/IGF-IIR) in preimplantation bovine embryos derived from different in vitro systems.

The aim of this study was to determine the relative abundance of mRNAs for the insulin-like growth factor I (IGF-I) and IGF-II ligands, and for the IGF receptors (IGF-IR and IGF-IIR) in in vitro preimplantation bovine embryos from the oocyte to the hatched blastocyst stage using two different culture systems: TCM-199 supplemented with oestrous cow serum, or synthetic oviduct fluid supplemented with polyvinyl alcohol. Development to the two- to four-cell stage and blastocyst stage was significantly higher (P < or = 0.05) in embryos cultured in TCM supplemented with oestrous cow serum than in those cultured in synthetic oviduct fluid supplemented with polyvinyl alcohol (61 and 25% versus 55 and 17%, respectively). A semi-quantitative RT-PCR assay did not detect IGF-I transcripts at any stage of preimplantation bovine development, including the hatched blastocyst stage. In both culture systems, IGF-IR, IGF-II and IGF-IIR were expressed throughout preimplantation development up to the hatched blastocyst stage in a varying pattern. The expression patterns of IGF-IR, IGF-II and IGF-IIR in embryos generated in the two culture systems were not significantly different, except at the expanded blastocyst stage, at which significantly higher amounts of IGF-IIR were observed in the TCM system than in the synthetic oviduct fluid system. These results indicate that transcripts of IGF-IR and IGF-IIR follow the standard pattern in which maternal stores of mRNA in the oocyte are slowly depleted up to the 16-cell stage and then re-established at the onset of embryonic expression of these genes. The lack of detectable IGF-I transcripts in the bovine embryo indicates a predominantly paracrine mode of action. The bovine embryo is capable of producing IGF-II, IGF-IIR and IGF-IR in large amounts, particularly after hatching, which may be important for the formation of the filamentous conceptus. Results indicate an autocrine mechanism for IGF-II and modulation of IGF family expression by culture conditions.

Analysis of Variance↗

PubMed2001

Current status and perspectives for the generation of transgenic pigs for xenotransplantation.

Xenotransplantation implies transplantation of organs between discordant, e.g. non-related species. This procedure usually is associated with a hyperacute rejection response (HAR) which destroys the transplanted organ within minutes. To overcome the growing shortage of human organs, transgenic pigs have been generated that express human complement regulatory genes. This approach enables to overcome the HAR as shown by an extended average survival rate of 40-90 days of the immunosuppressed primate recipient of a transgenic porcine heart. It is expected that transgenic pigs will be available as organ donors within the next 5-7 years. A major prerequisite is the prevention of the potential transfer of pathogenic microorganisms, in particular porcine endogenous retroviruses (PERV). Transgenic livestock has been generated predominantly via microinjection of DNA-constructs into pronuclei of zygotes. However, efficiency is low and only 1-3% transgenic offspring are to be obtained. Integration of the transgene occurs at random and expression is independent from the number of integrated copies but can be affected by the integration site. Improvements of the efficiency in the generation of transgenic pigs will be achieved by the use of genetically modified donor cells in nuclear transfer technology (cloning).

Animals↗